Fluorescence detection is rapidly gaining acceptance as the detection method of choice in a growing number of laboratory procedures. These include for example, automated DNA sequencing and a variety of immunoassays. In response to excitation, fluorescent dyes emit light at characteristic wavelengths which differ from the excitation wavelength. Multiple labels can be discriminated in a sample by selecting dyes which have distinguishable emission wavelengths. While fluorescence detection is able to rapidly discriminate small quantities of multiple labels, fluorescence detection scanners have generally been limited to qualitative determination of the location and relative concentrations of the labels.
Fluorescent labels are generally detected by directing an excitation beam at the label and collecting the resulting fluorescent emission. The intensity of the excitation beam is much greater than the intensity of the fluorescent emission produced by the excited label. Consequently, scattered excitation light which reaches the detector results in a high light background which significantly decreases the sensitivity of the instrument and interferes with quantitation of the labels.
A variety of electrophoresis instruments with integrated fluorescence detection systems are known. U.S. Pat. Nos. 4,971,677, 5,051,162 and 5,062,942, Kambara et al., describe systems in which excitation beams are directed at the side edges of electrophoretic gels, transverse to the electrophoretic pathways. Two-dimensional fixed detectors are positioned normal to the excitation beam path and located beneath, or to the side of, the gels.
Hunkapiller U.S. Pat. No. 4,811,218, et al., describes a real time scanning electrophoresis instrument in which a fixed location in the electrophoretic pathways is repetitively scanned by a moving excitation beam and detector. One of four interchangeable bandpass filters is positioned in the detection beam path of a collector lens for each sequential scan. A Fabry lens group, which images the collector lens, is located between the bandpass filters and a photomultiplier. The excitation beam is diverted toward the gel by a Brewster angle mirror to minimize polarized light scatter that interferes with fluorescence detection. The detection beam path is normal to the gel surface at a fixed angle relative to the excitation beam. The detector and Brewster angle mirror are fixed to a stage which moves back and forth across the gel to scan individual lanes.
U.S. Pat. No. 4,833,332, Robertson, Jr. et al., describes a scanning fluorescent detection system having dual detectors with complementary wavelength-selective filters. Multiple fluorophores having closely spaced overlapping emission spectra are discriminated by the ratio of the two detector outputs. An excitation beam is swept across the electrophoresis gel in a direction transverse to the electrophoretic pathways by a rotating mirror. The detectors are located on either side of the plane in which the excitation beam travels. The excitation beam plane is normal to the gel surface and the detectors simultaneously receive inputs from all points of the scanning path. A transmission filter, which rejects light having an angle of incidence less than 69 degrees, is placed between the wavelength selective filters and the gel to eliminate scattered excitation light and emitted fluorescence which would otherwise pass through the wavelength selective filters independent of the specified filter characteristics.
Two-dimensional scanners for post-separation fluorescence detection are also known. Laid-open European patent application 0 459 278 A1, describes a fluorescence pattern reading apparatus in which a post-separation electrophoresis gel or transfer membrane is moved past an excitation beam which sweeps a scanning path transverse to the sample's direction of motion. The scanning plane of the excitation beam is normal to the sample surface and a single detector is located adjacent to the scanning plane. A lens is placed between the sample and the detector's light collector to focus back-scattered excitation light, from the surface of the gel support, to a location separate from the light collecting surface of the detector. A pair of lenses separated by a diaphragm are placed between the light collector and a photomultiplier to extract the parallel light components of the inputted fluorescence. The parallel light components are directed to an optical filter which removes the components of scattered light and then focused on the photomultiplier by a third lens.
U.S. Pat. No. 4,877,966, Tomei et al., describes an apparatus for measuring low-level laser-induced fluorescence in tissue samples in which an optical detector is placed on the opposite side of the target from the excitation beam. A bias-cut fiberoptic face plate is positioned between the target and detector to reject the excitation light, which is normal to the face plate surface.
A large portion of the interfering excitation-light background arises from light scattering at the intersection of the incident excitation beam and sample support. Prior art attempts to reduce the interfering excitation-light background have done so at the cost of attenuating the fluorescence emission signal. Systems employing lenses in the light detection path suffer from the inherent optical inefficiency of lenses. Optically efficient systems, employing fiberoptic collectors or filters, generally have light collecting surfaces which reject a portion of the fluorescence emission as well as the scattered excitation light.
It is therefore an object of the present invention to provide an improved two-dimensional fluorescence detection scanner capable of selectively reducing excitation-light background without attenuating the fluorescence emission signal.